Despite its extraordinary effectiveness against smallpox infection, the parameters of protective immunity generated by vaccinia remain incompletely understood. Large numbers of individuals are at high risk for serious toxicities if vaccinated with or exposed to vaccinia, and the need to develop safer vaccines against smallpox underscores the importance of understanding the immunologic basis for such protection. This project will undertake a detailed examination of T and B cell responses to immunization with vaccinia and MVA under carefully controlled conditions in normal subjects, and to MVA in patients with atopic dermatitis or patients with hematologic malignancies and immune deficiency after hematopoietic stem cell transplantation. The studies will measure neutralizing and other binding antibodies, lymphoproliferative responses by 3H Thymidine incorporation, cytotoxic T-cell activity by a 51Cr release assay, and T-cell gamma interferon responses by ELISPOT. Normal subjects who were vaccinated with either vaccinia or MVA, will be challenged with vaccinia to determine whether protection against challenge was conferred. Measurements of immune responses will be conducted sequentially at multiple time points, including those before and after challenge. Rates and patterns of responses wilt be compared between vaccinia and MVA recipients, and between normals and the patient cohorts. The specificity of the B and T cell responses will be examined employing bioinformatics approaches and molecular techniques of the Research Resource Technical Development Component of the Center. These studies will measure antibody responses to a panel of recombinant vaccinia-encoded proteins identified by bioinformatics as potentially important in neutralization, including the EGF-like domain of DIL (SPGF). Since all subjects in our initial studies will be HLA-A*0201 positive, we will be able to utilize peptides from the recombinant vaccinia proteins to characterize class I allele specific T-cell responses by ELISPOT, cytokine flow cytometry, and peptide-MHC tetramers. Non-class I allele specific T-cell responses will be examined using whole recombinant proteins. Spectratype analysis will also be used to identify predominant T-cell epitopes. These studies wilt provide a comprehensive picture of the immune responses to vaccination with vaccinia and MVA, and identify epitopes against which important responses are directed. These studies will also demonstrate whether 2 groups of high-risk patients respond to MVA in a similar fashion to normals. The proposed studies in years 1 and 2 focus on MVA, but similar approaches would be utilized to study other candidate vaccines against smallpox, as well as vaccines against other agents important in biodefense.